Favorable clinical response and drug retention of anti-IL-6 receptor inhibitor in rheumatoid arthritis with high CRP levels: the ANSWER cohort study.

OBJECTIVE: Currently, biological disease-modifying anti-rheumatic drugs (bDMARDs) with different modes of action [tumour necrosis factor inhibitor (TNFi), interleukin-6 receptor inhibitor (IL-6Ri), or cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig)] are used in clinical practice to treat rheumatoid arthritis (RA). However, it is unclear which type of bDMARD is the most efficacious for a specific clinical situation. C-reactive protein (CRP) is an acute-phase reactant driven by IL-6 signalling. Here, we aimed to establish whether therapeutic efficacy differs between IL-6Ri and other bDMARDs with alternative modes of action in RA patients according to their CRP level. METHOD: RA patients treated with bDMARDs were enrolled from an observational multicentre registry in Japan. Patients were classified into three groups according to baseline CRP tertiles. The overall 3 year retention rates of each bDMARD category were assessed. The Clinical Disease Activity Index (CDAI) was also assessed before and 3, 6, and 12 months after bDMARD initiation. RESULTS: A total of 1438 RA patients were included and classified into three groups according to tertiles of baseline CRP levels (CRP1, 0-0.3; CRP2, 0.3-1.8; CRP3, 1.8-18.4 mg/dL). In CRP3, the overall 3 year drug retention rates were significantly higher for IL-6Ri than for TNFi and CTLA4-Ig (77.5 vs 48.2 vs 67.3, respectively). No significant difference was evident in terms of CDAI 12 months after bDMARD initiation in CRP1-CRP3. CONCLUSION: IL-6Ri may be a favourable therapeutic option over TNFi and CTLA4-Ig in RA patients with high CRP levels.

Brain-computer interface with rapid serial multimodal presentation using artificial facial images and voice.

Electroencephalography (EEG) signals elicited by multimodal stimuli can drive brain-computer interfaces (BCIs), and research has demonstrated that visual and auditory stimuli can be employed simultaneously to improve BCI performance. However, no studies have investigated the effect of multimodal stimuli in rapid serial visual presentation (RSVP) BCIs. The present study proposed a rapid serial multimodal presentation (RSMP) BCI that incorporates artificial facial images and artificial voice stimuli. To clarify the effect of audiovisual stimuli on the RSMP BCI, scrambled images and masked sounds were applied instead of visual and auditory stimuli, respectively. The findings indicated that the audiovisual stimuli improved performance of the RSMP BCI, and that P300 at Pz contributed to classification accuracy. Online accuracy of the BCI reached 85.7 ± 11.5 %. Taken together, these findings may aid in the development of better gaze-independent BCI systems.

Treatment with protein kinase C activator is effective for improvement of male pronucleus formation and further embryonic development of sperm-injected oocytes in pigs.

To assist the process of oocyte activation, which is essential for promotion of fertilization events, i.e., resumption of meiosis, extrusion of the second polar body and formation of the pronucleus (PN), artificial stimuli such as an electrical pulse have been applied to porcine oocytes after injection of sperm. However, the efficiency of fertilization and embryonic development remains low. It is well known that in vertebrates, inactivation of mitogen-activated protein (MAP) kinase is required for oocyte activation. We have hypothesized that even after electrical stimulation of sperm-injected oocytes, MAP kinase may not be inactivated. As it has been reported that MAP kinase activity is regulated by protein kinase C, we examined the effectiveness of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for improvement of fertilization and embryonic development of sperm-injected porcine oocytes. First, we examined the concentrations (0, 0.01, 0.1, 1, and 10 µM) and durations (0, 1, 3, 5 hours) of PMA treatment that were efficient for the extrusion of two polar bodies and formation of two PNs (2PB+2PN) and embryonic development. When the sperm-injected oocytes were treated with 0.01-µM PMA for 3 hours after electrical stimulation, the rates of 2PB+2PN and embryonic development were higher than those in the other treatment groups. We then examined the effect of PMA treatment (0.01 µM, 3 hours) on MAP kinase activity. Unexpectedly, after electrical stimulation, the activity remained low until PN formation, irrespective of whether or not the oocytes had been treated with PMA. On the other hand, transformation of the injected sperm nucleus into the male PN was accelerated after the PMA treatment. Our present results suggest that the low efficiency of fertilization and embryonic development in sperm-injected oocytes is not due to high activity of MAP kinase but due to poor transformation of the injected sperm nucleus into the male PN. Furthermore, a combination of electrical stimulation and PMA is a fairly effective artificial protocol for promoting 2PB+2PN and embryonic development in sperm-injected porcine oocytes.

Porcine androgenetic embryos develop to fetal stage in recipient mothers.

In livestock, parthenogenic embryos are simple to produce, but androgenetic embryos have been successfully produced only in sheep and cows. In the present study, matured porcine oocytes were enucleated by micromanipulation and then fertilized with sperm in vitro, thereby producing porcine androgenetic embryos. Porcine androgenetic embryos, which had only sperm genomes, were assessed for cleavage and for blastocyst formation 2 and 6 d after IVF, respectively. There was no difference in cleavage rate between androgenetic embryos and biparental IVF embryos (mean ± SD androgenetic: 65.5 ± 5.4%; biparental IVF: 63.2 ± 3.6%), but there was a difference in the rate of blastocyst formation (androgenetic: 4.5 ± 0.7%; biparental IVF: 30.2 ± 2.6%, P < 0.05). The average number of cells in Day 6 androgenetic blastocysts (34.3 ± 18.2) was lower (P < 0.05) than that in biparental IVF blastocysts (44.1 ± 19.5), but did not differ from that in parthenogenetic embryos (35.7 ± 16.7). The androgenetic embryos were transferred into recipient mothers to examine the competence of post-implantation development. Androgenetic fetuses were present on Days 21 and 25, but not on Days 28, 31, or 35. Of the six androgenetic fetuses recovered on Day 21, five had normal, translucent bodies, and two of these five had beating hearts. The four fetuses recovered on Day 25 were all non-viable. In conclusion, porcine androgenetic embryos initiated embryogenesis and had reached a viable fetal stage 21 days after IVF.

Interleukin-10 expression induced by adeno-associated virus vector suppresses proteinuria in Zucker obese rats.

Varying degrees of metabolic abnormalities mediated by chronic inflammation are implicated in the chronic glomerular injuries associated with obesity. Interleukin (IL)-10, a pleiotropic cytokine, exerts anti-inflammatory effects in numerous biological settings. In the present study, we explored the biological benefits of adeno-associated virus (AAV) vector-mediated sustained IL-10 expression against the pathological renal characteristics observed in Zucker fatty rats (ZFRs). We injected an AAV vector, encoding rat IL-10 or enhanced green fluorescent protein (GFP) into male ZFRs at 5 weeks of age. Subsequently, the renal pathophysiological changes were analyzed. Persistent IL-10 expression significantly reduced the urinary protein excretion of ZFRs compared with GFP expression (47.1±11.6 mg per mg·creatinine versus 88.8±30.0 mg per mg·creatinine, P<0.01). The serum levels of IL-10 negatively correlated with the urinary protein in AAV-treated rats (r=-0.78, P<0.01). Renal hypertrophy, increased widths in the glomerular basement membrane, and the lack of uniformity and regularity of the foot process of the visceral glomerular epithelial cells of ZFRs were significantly blunted by IL-10 expression. IL-10 also abrogated the downregulation of glomerular nephrin observed in ZFRs treated with the GFP vector. Our findings provide insights into the potential benefit of the anti-inflammatory effects of IL-10 on the overall management of glomerulopathy induced by the metabolic disorders associated with obesity.

A simple method for producing tetraploid porcine parthenogenetic embryos.

The objective was to produce porcine tetraploid parthenogenetic embryos using cytochalasin B, which inhibits polar body extrusion. Porcine cumulus-enclosed oocytes aspirated from antral follicles were cultured for 51 h, and treated with cytochalasin B from 35 h to 42 h after the start of culture. After maturation culture, 74.7% (2074/2775) of oocytes treated with cytochalasin B did not extrude a polar body (0PB oocytes). In contrast, 80.4% (1931/2403) of control oocytes extruded a polar body (1PB oocytes). The 0PB oocytes were electrically stimulated, treated with cytochalasin B again for 3 h, and then cultured without cytochalasin B. Six days after electrical stimulation, 49.8% (321/644) reached the blastocyst stage. The number of cells in these blastocysts derived from 0PB oocytes was significantly lower than that from 1PB oocytes (0PB: 24.9 ± 10.6; 1PB: 43.0 ± 17.1; mean ± SD). A porcine chromosome 1-specific sequence was detected in parthenogenetic 0PB embryos by fluorescence in situ hybridization (FISH) analysis. Typical pronucleus-stage samples derived from 0PB embryos had two pronuclei, each with two signals. In two-cell and blastocyst-stage embryos, four signals were detected in each nucleus derived from 0PB embryos. We inferred that 0PB oocytes, which had a tetraploid number of chromosomes, started to develop as tetraploid parthenotes after electrical stimulation, and that tetraploid status was stably maintained during early embryonic development, at least until the blastocyst stage.

Pseudoaldosteronism with increased serum cortisol associated with pneumonia, hypouricemia, hypocalcemia, and hypophosphatemia.

We describe here the interesting case of a 73-year-old hypertensive man with pseudoaldosteronism. He had been taking glycyrrhizin at a dose of 75 mg/day for 12 years because of mild liver damage, but had never experienced any previous symptoms associated with hypokalemia. He was referred to our hospital because of hypokalemic tetraparesis and rhabdomyolysis. At that time, we noted mineralocorticoid excess characterized by hypokalemia due to urinary K loss, exacerbation of hypertension due to increased tubular Na reabsorption, metabolic alkalosis, and suppression of both plasma renin activity and plasma aldosterone concentration. His urinary free cortisol excretion rate and the urinary ratio of free cortisol to free cortisone were markedly elevated. Thus we diagnosed pseudoaldosteronism that was related to the long-term use of glycyrrhizin. When he developed pseudoaldosteronism, he also contracted pneumonia, and exhibited elevated levels of serum cortisol and creatinine clearance (CCr) as well as hypouricemia, hypocalcemia, and hypophosphatemia. All normalized after the recovery from pneumonia and the administration of spironolactone. The extracellular volume expansion associated with increased tubular Na reabsorption by the aldosterone-sensitive distal nephron and the resulting increase in CCr caused an inhibition of proximal tubular reabsorption of uric acid, Ca, and inorganic phosphate, leading to their renal loss and therefore hypouricemia, hypocalcemia, and hypophosphatemia, respectively. In this patient, the increased circulating cortisol associated with the stress of inflammation caused by pneumonia triggered the development of pseudoaldosteronism.

Effects of oxygen tension and follicle cells on maturation and fertilization of porcine oocytes during in vitro culture in follicular fluid.

The objective was to investigate the effects of oxygen tension and follicle cells (FCs) during in vitro maturation of porcine oocytes in only porcine (Sus scrofa domesticus) follicular fluid (pFF), using static and non-static (rotating) culture systems, on the nuclear maturation and subsequent in vitro fertilization of the oocytes. In the first experiment, cumulus-oocyte complexes (COCs) were matured for 48 h in pFF supplemented with (+) or without (-) FCs (5.2x10(6) cells/mL), using the static (S) and rotating (R) culture systems (+FC/S, -FC/S, +FC/R, and -FC/R) under 5% or 20% O(2). Co-culture with FCs in the static culture system (+FC/S) had a detrimental effect on the meiotic competence of oocytes, whereas co-culture with FCs in the rotating culture system (+FC/R) increased maturation rates. In both culture systems, oxygen tension had no apparent effects on meiotic competence of oocytes, irrespective of culture system and FC addition. In the second experiment, COCs were matured under 5% or 20% O(2) using the -FC/S or +FC/R culture systems and then fertilized. Oxygen tension had no significant effects on fertilization parameters, irrespective of the culture system. The rotating culture system increased rates of sperm penetration and male pronuclear formation and decreased polyspermic fertilization compared with the static culture system (P < 0.05). In conclusion, both -FC/S and +FC/R culture systems supported meiotic competence, irrespective of oxygen tension. However, the +FC/R culture system may be superior to the -FC/S culture system for promoting fertilization.

Identification of genes downregulated during differentiation of porcine mesenteric adipocytes.

Adipose tissue development is a process that comprises not only hypertrophy, but also hyperplasia, of adipocytes. Although the proliferation of undifferentiated preadipocytes plays an important part in hyperplasia, this process is less well understood than the post-proliferation differentiation process. Despite the potential importance of porcine visceral adipose tissue to both meat production and biomedical research, there has been little study of this tissue and, in particular, its development and differentiation. To detect the genes involved in the maintenance of porcine visceral preadipocytes in an undifferentiated state or in the inhibition of adipocyte differentiation, we performed suppression subtractive hybridization using mesenteric preadipocytes in which fragments of the genes that are downregulated at 2 d of differentiation were enriched. We selected 672 clones and subjected them to differential screening and semiquantitative reverse transcription (RT)-PCR. As a result, we identified 34 downregulated genes. Among these, the detailed expression patterns of 6 genes were examined using real-time RT-PCR in both preadipocytes during in vitro differentiation and cell fractions directly isolated from pig mesenteric adipose tissue. The expressions of connective tissue growth factor, AXL receptor tyrosine kinase, stromal membrane-associated protein 1-like, and retinoic acid-induced 14 were significantly downregulated during adipocyte differentiation in vitro (P < 0.05), and the expressions of Rho/Rac guanine nucleotide exchange factor 2 and secreted frizzled-related protein 4 also tended to be decreased, although not significantly. Furthermore, all 6 genes showed significantly greater expression in stromal vascular cells, which contain preadipocytes, than in mature adipocytes (P < 0.05), raising the possibility that these genes are involved in adipocyte differentiation in vivo as well as in vitro.

Growth inhibition of human salivary gland tumor cells by introduction of progesterone (Pg) receptor and Pg treatment.

Cancer of the salivary gland is one of the common cancers in the head and the neck regions. This type of cancer develops in the minor and the major salivary glands, and it sometimes metastasizes to other organs, particularly the lung. Morphologic mimicry and similarity in the expression of steroid hormone receptors between salivary gland tumors (SGTs) and breast tumors are well-known phenomena and are occasionally debated in the field of surgical pathology. Progesterone (Pg), one of the female sex steroid hormone, is intimately involved in the development of the mammary gland. Further, it is believed that Pg plays a role in breast cancer progression. However, little is known regarding its role in SGTs. In this study, we used ACCM, a human adenoid cystic carcinoma cell line established from the salivary gland, in order to clarify the role of the Pg receptor (PR) on cell proliferation. No effect of Pg on cell proliferation was observed in the PR-deficient aggressive ACCM cells. However, after introducing PR into the ACCM cells, Pg markedly inhibited the proliferative activity of the cells. This inhibitory effect on cell proliferation was accompanied by p21 upregulation, and Id1 and c-myc downregulation. Moreover, Pg-treated PR transfectants showed significant morphological change; they appeared more flattened and spread out when compared with the ethanol-treated control cells. Our results provided significant insights into the mechanism of suppression of the proliferative property of the cells via the function of PR, and suggested that PR reintroduction therapy might be a viable method of inhibiting human SGT progression.

Intraoperative real-time genetic diagnosis for sentinel node navigation surgery.

Sentinel node navigation surgery (SNNS) has received considerable attention for its role in deciding whether to perform neck dissection in patients with early oral cancer. However, diagnostic accuracy and its intraoperative availability of results remain important concerns. First, we shortened the examination time required for genetic diagnosis. Second, we assessed the quality of the extracted mRNA. Third, 10 patients with early N0 oral cancer underwent SNNS, using our new technique for genetic diagnosis to determine whether neck dissection was required. The examination time of our one-step reverse-transcriptase polymerase chain reaction method using a minicolumn and LightCycler was successfully shortened to 2 h, permitting intraoperative genetic diagnosis. The extracted mRNA was of high quality. Six sentinel nodes in four patients were diagnosed to be metastatic on genetic diagnosis; these patients underwent neck dissection. The other six patients avoided unnecessary surgery. We conclude that intraoperative genetic diagnosis of micrometastasis holds promise of being a sensitive method that can be used to support SNNS.

The effects of GH-releasing hormone/somatostatin on the 5'-promoter activity of the GH gene in vitro.

The two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIF), are known to regulate GH secretion. However, the effects of these hormones on GH gene expression are not completely clear, partly because of the lack of appropriate host cells maintaining the original characteristics of the somatotroph. Since MtT/S, a pure somatotroph cell line, has become available, the effects of GHRH and SRIF on GH gene transcription have been studied using a subclone of MtT/S (MtT/SGL), in which the GH gene 5'-promoter-luciferase fusion gene was stably incorporated. The expression of GHRH receptor and SRIF receptor subtypes was also studied by RT-PCR. The results showed that MtT/SGL cells intrinsically expressed the functional GHRH receptor and all of the SRIF receptor subtypes. The expression of GHRH receptor was markedly enhanced by glucocorticoid pretreatment and, in the presence of corticosterone and 3-isobutyl-1-methylxanthine, GHRH (at or above 100 pM) stimulated GH gene 5'-promoter activity in a dose-dependent manner. On the other hand, SRIF (100 nM) significantly antagonized the effect of GHRH, which was completely reversed by pretreatment with pertussis toxin (50 ng/ml). Taken together, the present data indicated that both GHRH and SRIF are involved in the transcriptional regulation of the GH gene, and that the effect of SRIF is mediated through pertussis toxin-sensitive G protein. The MtT/SGL cell line is a good in vitro model for studying the molecular mechanisms of GH gene transcription by GHRH and/or SRIF.

Assignment of 64 genes expressed in 28-day-old pig embryo to radiation hybrid map.

A swine resource family was constructed at the National Institute of Animal Industry, Japan, in order to determine the genetic regions responsible for economically important traits, including fetus development. To identify genes expressed in the early stage of embryo development, we cataloged and mapped genes expressed in a 28-day-old normal pig embryo. In this effort, we have mapped 64 genes, which have map information in human genome onto a swine radiation hybrid (RH) map, IMpRH. These mappings provided additional chromosomal homologies between swine and human to improve the comparative map between the two species. The distribution of the genes assigned to swine chromosomes are as follows: 9 genes were assigned on SSC6; 6 genes each assigned on SSC5 and SSC14; 5 genes each assigned on SSC3, SSC4, and SSC8; 4 genes each assigned on SSC1, SSC7, SSC9, and SSC15; 3 genes each assigned on SSC2, SSC13 and SSCX; and 1 gene each assigned on SSC10, SSC11, and SSC16. Moreover, the present findings revealed 18 new chromosomal homologies between pig and human. Briefly, SSC3 regions were indicated to correspond with HSA1 and HSA10; SSC4 with HSA6; SSC5 with HSA2, HSA15, and HSA16; SSC6 with HSA3, HSA6, and HSA20; SSC7 with HSA11; SSC8 with HSA3, HSA6, and HSA7; SSC9 with HSA8; SSC13 with HSA1; SSC14 with HSA13; SSC15 with HSA19; SSC16 with HSA9.

Electroretinogram analysis of relative spectral sensitivity in genetically identified dichromatic macaques.

The retinas of macaque monkeys usually contain three types of photopigment, providing them with trichromatic color vision homologous to that of humans. However, we recently used molecular genetic analysis to identify several macaques with a dichromatic genotype. The affected X chromosome of these animals contains a hybrid gene of long-wavelength-sensitive (L) and middle-wavelength-sensitive (M) photopigments instead of separate genes encoding L and M photopigments. The product of the hybrid gene exhibits a spectral sensitivity close to that of M photopigment; consequently, male monkeys carrying the hybrid gene are genetic protanopes, effectively lacking L photopigment. In the present study, we assessed retinal expression of L photopigment in monkeys carrying the hybrid gene. The relative sensitivities to middle-wavelength (green) and long-wavelength (red) light were measured by electroretinogram flicker photometry. We found the sensitivity to red light to be extremely low in protanopic male monkeys compared with monkeys with the normal genotype. In female heterozygotes, sensitivity to red light was intermediate between the genetic protanopes and normal monkeys. Decreased sensitivity to long wavelengths was thus consistent with genetic loss of L photopigment.

Keratin mRNA for detecting micrometastasis in cervical lymph nodes of oral cancer.

We studied three keratin (K) gene candidates, K13, K19, and K20 mRNAs, for detecting micrometastases in cervical lymph nodes (LNs) by reverse transcriptase-polymerase chain reaction (RT-PCR). Of 166 histologically metastasis-negative nodes, 24 micrometastatic LNs (14. 4%) were detected based on K13 gene expression. Keratin 19 mRNA is an inadequate marker for the genetic diagnosis due to not only illegitimate gene expression from lymphatic tissue but also gene expression from the ectopic salivary gland. Keratin 20 mRNA showed low sensitivity. It is suggested that K13 mRNA may be a promising tumor marker among these keratin genes for detecting the micrometastases in cervical LNs of oral cancer.

Gene expression of GLUT isoforms and VHL in oral squamous cell carcinoma.

In human oral squamous cell carcinoma (OSCC) cell lines, we detected atypical mRNA expression of GLUT2 and/or GLUT4 in addition to enhanced expression of GLUT1 mRNA using RT-PCR. In semi-quantitative reverse transcription-polymerase chain reaction analysis of mRNA expression in OSCC cell lines, we found an inverse relationship between mRNA expression of von Hippel-Lindau (VHL) and that of GLUT1, with no apparent influence on the expression of other GLUTs. These findings suggest that the reduction of VHL may play a critical role in glucose uptake of OSCC cell lines, with enhancement of GLUT1 expression.

Pig cloning by microinjection of fetal fibroblast nuclei.

Pig cloning will have a marked impact on the optimization of meat production and xenotransplantation. To clone pigs from differentiated cells, we microinjected the nuclei of porcine (Sus scrofa) fetal fibroblasts into enucleated oocytes, and development was induced by electroactivation. The transfer of 110 cloned embryos to four surrogate mothers produced an apparently normal female piglet. The clonal provenance of the piglet was indicated by her coat color and confirmed by DNA microsatellite analysis.

Replicative advantage and tissue-specific segregation of RR mitochondrial DNA between C57BL/6 and RR heteroplasmic mice.

To investigate the interactions between mtDNA and nuclear genomes, we produced heteroplasmic maternal lineages by transferring the cytoplasts between the embryos of two mouse strains, C57BL/6 (B6) and RR. A total of 43 different nucleotides exist in the displacement-loop (D-loop) region of mtDNA between B6 and RR. Heteroplasmic embryos were reconstructed by electrofusion using a blastomere from a two-cell stage embryo of one strain and an enucleated blastomere from a two-cell stage embryo of the other strain. Equivalent volumes of both types of mtDNAs were detected in blastocyst stage embryos. However, the mtDNA from the RR strain became biased in the progeny, regardless of the source of the nuclear genome. The RR mtDNA population was very high in most of the tissues examined but was relatively low in the brain and the heart. An age-related increase of RR mtDNA was also observed in the blood. The RR mtDNAs in the reconstructed embryos and in the embryos collected from heteroplasmic mice showed a different segregation pattern during early embryonic development. These results suggest that the RR mtDNA has a replicative advantage over B6 mtDNA during embryonic development and differentiation, regardless of the type of nuclear genome.